The following describes certain relevant art, none of which is admitted to be prior art to the appended claims.
Various methodologies are available for the visualization of cells or molecules in cells and for the measurement of analyte concentrations in fluids. Fluorescence microscopy utilizes fluorescent dyes, generally connected to specific probes, such as antibodies, for the localization of proteins and complexes in cells.
For the measurement of analyte concentrations, detection of an analyte of interest, determination of the particular sequence of a nucleic acid molecule, immunoassays and various hybridization methods have become popular over the last 40 years. Radioimmunoassays were developed because the high specific activity of the radionucleotide allowed measurement of very low concentrations of analyte. However, because of the concerns for the environment and human health, the use of radionucleotides in immunoassays is becoming less popular. The use of enzymes in immunoassays to amplify a signal has been a very important advance in the field of immunoassays because their use does not involve environmental or human health hazards or risks. Enzyme-linked immunoassays, however, can be problematic because the activity of the enzyme is temperature dependent and the instability of the enzyme or the substrates can result in inaccurate quantitation of the target ligand. Still other immunoassays monitor fluorescence as the signal, with or without enzymes, for the measurement of analyte concentrations.
Bi-fluorophore energy transfer dyes have been described which provide a novel methodology for monitoring processes in biological systems. The fluorescent nature of such dyes enables them to monitor processes in which the biological systems are involved. The fluorescent signal is measured by a fluorometer which is tuned to excite the fluorescent molecule at a specific wavelength and to measure the emission of fluorescence at another wavelength. The difference in the excitation and emission wavelengths is referred to as the Stokes shift.
Previously, a variety of combinations of bi-fluorophore dyes have been described. U.S. Pat. No. 5,688,648, entitled “Probes Labelled with Energy Transfer Coupled Dyes” Mathies et al., filed Dec. 19, 1995, which is incorporated herein by reference in it's entirety, including any drawings, discloses sets of fluorescent labels carrying pairs of donor and acceptor dye molecules wherein the labels can be attached to nucleic acid backbones for sequencing. Included is a method for identifying and detecting nucleic acids in a multi-nucleic acid mixture by using different fluorescent labels, wherein the fluorescent moieties are selected from families such as cyanine dyes or xanthenes. The fluorescent labels comprise pairs of fluorophores where one fluorophore donor has an emission spectra which overlaps the fluorophore acceptor's absorption so that there is energy transfer from the excited member to the other member of the pair.
UK Patent No. 2301 833 B entitled “Fluorescent Labelling Complexes with Large Stokes' Shifts Formed by Coupling Together Cyanine and Other Fluorochromes Capable of Resonance Energy Transfer” Waggoner et al., filed May 30, 1996, which is incorporated herein by reference in it's entirety, including any drawings, discloses complexes comprising a first fluorochrome having first absorption and emission spectra and a second fluorochrome having second absorption and emission spectra. The linker groups between the fluorochromes are alkyl chains. The fluorescent nature of the dyes enables them to be of use in sequencing and in nucleic acid detection.
In bi-fluorophore dye construction, aspects of particular importance are the distance between the acceptor and donor molecules, and the structure of the linker groups. There remains a need for additional improvements in dye construction, for example in order to accommodate selection of biological molecules of various sizes.